This application bases priority on corresponding Hungarian application P94-02511, filed Aug. 31, 1994.
This invention relates to a diagnostic assay for the detection of multi-drug resistance.
Treatment of many diseases can be severely limited if the patient develops resistance to a chosen therapeutic drug. For example, chemotherapy, while generally an effective treatment against human cancerous diseases, is hampered when a patient becomes resistant to the chemotherapeutic. In one special form of drug resistance, called "Multi-drug Resistance," the patient's cells become resistant not only to the particular chemotherapeutic, but to a wide range of structurally and functionally unrelated drugs simultaneously (see Ford et al., Pharmacological Reviews, 42:155-199, 1992).
Multi-drug resistance is caused by an integral glycoprotein in the plasma membrane of the targeted cell. The most studied of these proteins is variously called Multi-Drug-Resistance 1 protein (MDR1), P-glycoprotein (pleiotropic-glycoprotein), Pgp, or P-170. Multi-drug resistance is also caused by a related homologue of MDR1, called the Multidrug-resistance Related protein (MRP)(Grant et al. Cancer Res. 54:357-361, 1994). The MDR1 and MRP proteins function as multi-drug transporters, catalyzing an energy dependent outward transport of drugs from the cell. They strongly resemble prokaryotic and eukaryotic members of the ABC (ATP Binding Cassette) transporters, or traffic ATPases (see Endicott et al., Annu. Rev. Biochem. 58:137-171, 1989; Higgins, Annu. Rev. Cell. Biol. 8:67-113, 1992).
MDR1 naturally functions to, and is highly expressed in tissues normally responsible for, extruding toxic materials and waste-products from cells (e.g., lung, kidney, and liver) and secreting hydrophobic compounds from exocrine or endocrine glands (Gottesman et al., J. Biol. Chem. 263:12163-12166, 1988; Higgins, Annu. Rev. Cell. Biol. 8:67-113, 1992). Consistent with its natural function, MDR1 catalyses an ATP-dependent extrusion of various cytotoxic drugs from the cell, e.g., vinca alkaloids, anthracyclines, and other natural antibiotics, thereby maintaining their cellular level at a subtoxic concentration. When expressed by tumor cells, MDR1 expels cytotoxic chemotherapeutic agents, and thus allows the tumor cell to survive anticancer treatments even at high drug doses. At the same time "ordinary" cells, having no such extrusion mechanism, may receive a lethal drug exposure. Tumors developing from tissues normally expressing MDR1 often show primary drug resistance, while in other tumors secondary drug resistance can develop during chemotherapy. A variety of agents have been proposed to reverse multi-drug resistance: e.g., verapamil, quinidine, calmodulin inhibitors, phenothiazines, reserpine, or cyclosporin A (Gottesman et al. Annu. Rev. Biochem. 62:385-427,1993).
In clinical treatment of various cancerous diseases reliable detection of multi-drug resistance would be extremely helpful. Combination chemotherapy treatment protocols could be adjusted and drug-resistance reversing agents could be applied accordingly.
In MDR1-expressing cells a decreased uptake of cytotoxic drugs can be visualized by measuring the cellular accumulation or uptake of fluorescent compounds, e.g., anthracyclines (Herweijer et al., Cytometry 10:463-468, 1989), verapamil-derivatives (Lelong et al., Mol. Pharmacol. 40:490-494, 1991), Rhodamine 123 (Neyfakh, Exp. Cell Res. 174:168-174, 1988); and Fluo-3 (Wall et al. J. Natl. Cancer Inst. 83:206-207, 1991; Wall et al. Eur. J. Cancer 29:1024-1027, 1993), respectively. However, quantitating cellular uptake of these fluorescence dyes may be difficult. For example, rhodamine 123 is poorly retained by the cells and interacts with various intracellular compartments and organelles, producing a spectral shift and a change in fluorescence intensity. Similarly, measurement of the steady-state distribution of fluorescent drugs or drug-mimicking compounds is made uncertain by the low sensitivity of the assay and the quenching effect of DNA and/or other cellular components.